P1278: ACTIVATION OF THE NECROPTOSIS PATHWAY PLAYS A ROLE IN SYNERGISM WITH COMBINATION OF THE IAP ANTAGONIST, TOLINAPANT AND HYPOMETHYLATING AGENTS (HMA) IN IN VITRO MODELS OF T-CELL LYMPHOMA (TCL).
Authors: J. S. Manavalan, I. Pal, A. Pursley, G. Ward, T. Smyth, M. Sims, D. Feith, T. P. Loughran, O. A. O’Connor, E. Marchi
Journal: HemaSphere (2022)

Background: Peripheral T-cell lymphoma (PTCL) are a highly aggressive and heterogeneous group of non-Hodgkin lymphomas that often carry a poor prognosis with standard chemotherapy. Tolinapant is a potent, non-peptidomimetic antagonist of cIAP1, cIAP2 and XIAP. In an ongoing Phase 2 trial (NCT02503423), tolinapant has shown activity against highly pre-treated peripheral and cutaneous T-cell lymphoma. HMAs have also shown clinical responses in some subsets of PTCL. Both HMAs and IAP antagonists show immunomodulatory anti-cancer potential in pre-clinical studies. We have also shown that decitabine and tolinapant as single agent induced necroptosis in mouse TCL cell lines with the activation of the RIPK1/RIPK3/MLKL necroptosis pathway. In addition, re-expression of RIPK3 in CT26 cells by CRISPR increased lytic cell death on treatment with tolinapant indicating its essential role in the necroptosis pathway. Aims: We explored the sensitivity of a range of TCL lines to tolinapant, established the synergy coefficient and interrogated the role of necroptosis in the mechanism of synergy between tolinapant and drugs active against PTCL, the HMAs; azacytidine and decitabine Methods: A panel of 10 human TCL lines were tested in proliferation assays (CellTiterGlo) for sensitivity to tolinapant in the presence or absence of 10 ng/ml of TNFa. For combination studies, each drug was tested at the IC10, IC20 and IC40 concentrations, in the presence or absence of TNFa. Synergy coefficient was calculated using the Excess over Bliss model with SynergyFinder. Additionally, the on-target effects of the drugs were measured by analyzing levels of the IAPs, DNMTs and key apoptosis and necroptosis markers, such as PARP and RIPK3 by Western blotting. Results: TCL Lines demonstrate varying sensitivities to tolinapant with the most sensitive cell line, ALK+ ALCL SUP-M2, having an IC50 as low as 20 nM ± 1 nM while a resistant CTCL cell line HH had an IC50 of over 20 µM. In combination experiments using both cell lines, tolinapant plus azacytidine or decitabine displayed a very high degree of synergism; Average synergy score of 50.9 and 49.8 with azacytidine and 22.6 and 31.3 with decitabine for HH and SUP-M2, respectively. Of note, a high degree of synergism was also seen with concentrations 10 fold lower of azacytidine or 100 fold lower of decitabine, added daily (Figure 1). The combination of tolinapant and the HMAs led to a decrease in the levels of cIAP1 and DNMT3a in TCL lines, demonstrating on-target activity of tolinapant and the HMAs, respectively. In addition, both azacytidine and decitabine increased the levels of RIPK3 up to 3-4 fold and 6-8 fold respectively, in a concentration dependent manner at 72Hrs by western blot analysis, indicating activation of the necroptosis pathway. Image:Figure 1: 3D synergy plots showing average synergy scores for decitabine in combination with tolinapant in the CTCL line HH.Summary/Conclusion: Tolinapant has demonstrated varying cytotoxic effects against a range of TCL lines as a single agent. Tolinapant, in combination with the HMAs, azacytidine and decitabine, displays a very high degree of synergism, in vitro, even at very low concentrations of both HMA drugs. The HMAs were able to activate the necroptosis pathway, as shown by the increase in RIPK3. Thus, activation of the necroptosis pathway, is a possible mechanism for the high degree of synergism displayed when HMAs are used in combination with tolinapant in the TCL lines in vitro. These data provide the rationale to initiate clinical trials with the combination of tolinapant and decitabine.